Strong Correlation of B₂-Microglobulin (B₂-m) with Procalcitonin (PCT) in the Serum of Chronic Hemodialysis Patients: A Role for Infections in the Dialysis-Related Amyloidosis?

Abstract

Introduction. Infections trigger the activation of defensive cells capable to produce and release B₂-microglobulin (B₂-m). Procalcitonin (PCT), secreted by a wide range of human cells, included the aforementioned defensive cells, is generally considered a sensitive and specific marker of infection. In this prospective study, we examined the possibility that infections, as detected by increased levels of PCT, increase the serum levels of B₂-m in chronic hemodialysis (CHD) patients, possibly affecting the rate of progression of dialysis-related amyloidosis (DRA). Methods. For a period of four months, 76 CHD patients, 45 men/31 women, mean age 63 ± 15.7 years, with no residual renal function and in HD for 46 ± 50 months were studied bimonthly. Blood was drawn, at baseline T(0), two months T(2), and four months T(4), for measuring hematocrit (Ht), white blood cells (WBC), erythrocyte sedimentation rate (ESR), blood urea and serum creatinine, protein (albumin, globulin), C-reactive protein (CRP), and PCT και B₂-m. Any events (especially infections) in the preceding 10-day period were recorded. Results. At baseline, 100% of all B₂-m measurements were abnormal (>2.4 mg/L), 13.4% of PCT values were increased (>1.5 ng/mL), and 49.4% of CRP values exceeded the lower limit of 5 mg/L with no statistically significant differences between the results of the three periods of the study. Statistically significant, in all periods, was the linear positive correlation of B₂-m with PCT (T[0]: p < 0.001, T[2]: p < 0.004, T[4]: p < 0.001). Also, statistically significant (p < 0.005) was the positive correlation of B₂-m to HD vintage. Conclusions. In this study, the strong positive correlation of B₂-m to PCT probably signifies that the (mainly subclinical) infections increase B₂-m production in CHD patients intensifying the problem of HD-related amyloidosis.

Keywords:

• hemodialysis
• procalcitonin
• B₂-microglobulin

INTRODUCTION

Dialysis-related amyloidosis (DRA) is a serious complication affecting chronic hemodialysis (CHD) patients due to the severely decreased or totally abolished ability of the kidneys to degrade B₂-microglobulin (B₂-m), the removal of which depends entirely on the type of dialyzer, the method used, and the duration of HD session. B₂-m is continuously produced and secreted from all nucleated cells of the body, including the defensive cells (macrophages, polymorphonuclear neutrophils, lymphocytes, etc.), and its deposition in all tissues can cause carpal tunnel syndrome, bone cysts, arthropathy, and malfunction of extraskeletal organs.[1],[2] Although DRA has been well-known for many years, little is known about the rate of B₂-m production. It is quite reasonable to hypothesize that inflammatory conditions, such as infections, by activating the aforementioned defensive cells can modify (most probably increase) the rate of secretion and the serum levels of B₂-m. Infections are common and mostly subclinical[3],[4] in CHD, and procalcitonin (PCT), a secretory protein released by various types of human cells,[5],[6] is considered to be a sensitive and reliable marker for their detection.[7–9]

Our prospective study aimed to detect any correlation of B₂-m to PCT (as a marker of infections) in the serum of CHD and to C-reacting protein (CRP), a widely accepted marker of non-specific inflammatory reaction[10–12] in a parallel assessment of the influence to B₂-m serum levels.

METHODS

Patients: Hemodialysis

This is a single-center prospective study. No patients had residual renal function, and all were informed about the aim and methodology of the study and gave their consent. The study lasted four months, and patients were tested bimonthly (i.e., at baseline T[0], two months T[2], and four months T[4]).

All patients had well-functioning arteriovenous (AV) access or permanent venous catheter and were dialyzed thrice weekly. Their time on HD was at least six months, and duration of each dialysis session was four hours. We used low flux membranes with material (mostly polysulfone) and surface capable of offering eKt/V ≥ 1.1. During the study all dialysis conditions were kept constant (i.e., dialyzer, kind of access, blood or dialysate flow, duration of dialysis session), and no change in drug administration occurred.

Detailed records were kept for all events in the preceding 10 days of every assessment, with an emphasis on well-documented infections defined by the combination of fever >38°C, WBC ≥ 10000/mm³, and clinical signs of infection (e.g., a productive cough).

Laboratory Investigation

Just before the first HD session of the week and at baseline and two and four months, blood was drawn from the arterial line for the following assessments (228 blood samples): Ht, hemoglobin, WBC, platelets, erythrocyte sedimentation rate (ESR), blood urea and serum creatinine, total protein, albumin, CRP, PCT, and B₂-m.

CRP was measured by the high sensitivity immunonephelometric method (High Sensitivity C-Reactive Protein Flex reagent cartridge, DADE BEHRING, Newark, New Jersey, USA). For CHD patients, values accepted as normal were ≤5 mg/L.[13–15]

B₂-m was measured by immunoenzymometric assay (TOSOH, Tokyo, Japan) with a normal range of 1–2.4 mg/L.

PCT was measured by immunoluminometric method (normal value <0.5 ng/mL) with the use of monoclonal antibodies (BRAHMS Diagnostica, Berlin, Germany). Though some investigators[16–19] use the above cut-off value, we adopted the value of 1.5 ng/mL as the upper normal limit of PCT for CHD patients[20–22] to avoid trivial and insignificant increases.

Statistical Analysis

Results are expressed as mean (standard deviation) for normally distributed continuous variables, as median (interquartile range) for skewed variables, and as frequency (%) for categorical variables. Continuous variables were tested for normal distribution using the Kolmogorov-Smirnov test. Bivariate correlations were assessed by Spearman rank correlation coefficient. Differences among the three time points were evaluated by non-parametric Friedman test. Comparisons between groups within the same time point were studied with Student's t-test for normally distributed variables; otherwise, the Mann-Whitney U test was applied. Categorical data were compared by X²-test or Fisher exact test where appropriate. ANOVA was used to assess multiple correlations. Because indices of interest (PCT, B2-m, CRP) had a non-normal distribution, data were analyzed after logarithmic transformation. All analyses were performed with the statistical software package STATA version 8.0 (Stata Corp.).

RESULTS

A total of 76 CHD patients were enrolled. The mean age of study population was 63 years (SD = 15.7). Patient's characteristics and laboratory assessments at the three phases are presented in Tables 1 and 2. There were no differences in levels of B2-m, CRP, and PCT among the three time points (see Table 2). Less than 3% of the laboratory assessments were preceded by well-documented infection, which made the incorporation of this parameter in the statistical analysis impossible.

Table 1 Baseline patient's characteristics and laboratory assessments

Parameters	Median	IQR
Age (yrs)	66	56–75
Sex (male)^†	43(59)	—
Time on dialysis (months)	32	14–57
Weight (kg)	67.5	58–80
Urea (mg/dL)	178.5	153–205
Creatinine (mg/dL)	10.2	8.6–11.8
Hematocrit (%)	35.6	33.3–38.5
WBC (/mm^3)	7235	5700–8220
ESR (mm/h)	50	27–70
Total proteins (g/dL)	6.8	6.6–7.2
Serum albumin (g/dL)	3.4	3.3–3.6

Values at two and four months did not differ statistically significant from corresponding ones at baseline.

*IQR = interquartile range.

^†presented as N (%).

Table 2 Levels of B2-m, PCT and CRP at baseline T(0), two months T(2), and four months T(4)

		Time (months)			p
		T(0)	T(2)	T(4)
B2-m (mg/l)	Median	26.1	249	27	0.43
	IQR^†	21.8–29.8	19.2–29	21.6–41
PCT (ng/ml)	Median	0.9	0.9	0.9	0.07
	IQR^†	0.6–1.3	0.7–1.4	0.5–1.5
CRP (mg/l)	Median	5.06	8	5.27	0.63
	IQR^†	2.5–12	1.8–13	2.7–21

^*Friedman test.

^†IQR = interquartile range.

β₂-m

In all three phases of the study, values of β₂-m exceeded the upper normal limit of 2.4 mg/L. β₂-m and PCT concentrations were significant correlated at all point times (T[0]: r = 0.7, p < 0.001; T[2]: r = 0.3, p = 0.004; T[4]: r = 0.6, p < 0.001). Figure 1 illustrates β₂-m levels in the two groups of PCT (cut-off 1.5 ng/mL) at baseline. Linear relations were studied at baseline only (T0) after logarithmic transformations, as there were no differences in β₂-m and PCT among three time points. The β₂-m at baseline was significantly associated with PCT (F = 3.37, p < 0.001). After adjustment for age, sex, duration on HD, and CRP, the association between β₂-m and PCT levels remained unchanged.

Figure 1. Comparison (Mann-Whitney U test) of B₂-m levels in two groups of PCT (cut-off value 1.5 ng/mL) at baseline.

Between β₂-m and CRP levels, significant differences were not found at any time of the study (T[0]: r = 0.13, p = 0.233; T[2]: r = 0.11, p = 0.304; T[4]: r = 0.12, p = 0.321). Figure 2 illustrates β₂-m levels in two groups of CRP (cut-off 5mg/L) at baseline. After logarithmic transformations, β₂-m at baseline was not significantly associated with CRP levels (F = 1.53, p = 0.220).

Figure 2. Comparison (Mann-Whitney U test) of B₂-m levels in two groups of CRP (cut-off value 5 mg/L) at baseline.

Moreover, the β₂-m concentrations were positively correlated with dialysis vintage (T[0]: r = 0.3, p = 0.04; T[2]: r = 0.4, p = 0.001; T[4]: r = 0.4, p = 0.001). On the contrary, there was no significant correlation of β₂-m to Ht, WBC, albumin, and ESR.

PCT

At baseline, 13.4% of PCT concentrations were greater than 1.5 ng/mL in CHD patients. PCT levels correlated well to dialysis vintage. On the contrary, there was no correlation between PCT and albumin at any time (T[0]: p = 0.94; T[2)]: p = 0.69; T[4]: p = 0.52). The relationship of PCT to CRP was not statistically significant (T[0]: X² = 0.86, p = 0.35; T[2]: X² = 1.03, p = 0.31; T[4]: X² = 3.4, p = 0.06).

CRP

Baseline plasma CRP levels were over the upper normal limit of 5 mg/L in 49.4% of the population. CRP concentrations were negatively correlated to albumin (p < .001 at all three time points) and positively correlated to ESR (T[0]: r = 0.32, p = 0.007; T[2]: r = 0.5, p < 0.001; T[4]: r = 0.4, p = 0.002).

DISCUSSION

In this prospective study of CHD patients, a statistically significant positive and linear correlation was noticed between serum levels of B₂-microglobulin and PCT, suggesting a role for infections in the rate of progression of DRA. This interesting finding of our study, which to our knowledge has not been described in the literature, was still valid even after adjustment for age, sex, dialysis vintage, and CRP. On the contrary, there was no statistically significant correlation of B₂-m to CRP in all time periods.

Taking into account that PCT is considered a marker of infection,[7–9],[16],[17],[23] the correlation of B₂-m to PCT is most likely attributed to the expression and production of these two substances from activated cells (i.e., monocytes, macrophages, lymphocytes).[5],[17],[24],[25] This activation can be due to the secretion of cytokines and interferons during the immune response in the setting of pro-inflammatory substances release syndrome. Though this release can be provoked by infections or non-infectious conditions (e.g., contact of blood with bioincompatible membranes), the lack of correlation between B₂-m to CRP, an accepted marker of systemic inflammatory reaction,[10–12] in all time periods points to the simultaneous production and release of B₂-m and PCT only (or mostly) by infections. These infections must have been mostly subclinical, judged by the low percentage of referred clinically overt infections (3%) in our study. Considering the fact that infections are common in CHD patients, particularly with the frequent use of temporary or permanent vascular catheters, the strong correlation of B₂-m and PCT suggests that during infectious status, judged by increased PCT serum levels, B₂-m production and tissue accumulation is enhancing the rate of progression of DRA. The statistically significant correlation of both PCT and B₂-m to the dialysis vintage must be attributed to the increased, time-dependent production and secretion of cytokines due to blood contact with bio-incompatible parts of HD procedure and to the greater impact of infections that activate the aforementioned cells. In a similar study, Level et al.[18] found no correlation of PCT and dialysis vintage.

In contrast to the strong negative correlation of CRP to albumin, there was no correlation of PCT to albumin in our data. Reports from the literature are contradictory, as negative[18],[26] and a lack of any[19] correlation of PCT to albumin are both reported.

The higher percentage of abnormal CRP (considered as index of the broader term of systemic inflammatory reaction) values (49.4%) over PCT (13.4%) strongly suggests that PCT constitutes a more specific marker of infections than CRP. We must mention that increases of PCT are noticed in several non-infectious conditions, such as acute myocardial infarction, major surgery, burns, and shock, as well as in dialysis patients.[17],[19,], [26–28] Their influence on the results of our study must be small (if any exists at all), as most of these conditions are easily recognized and did not occur in our patients, and the referred increases are small (mostly less than the defined 1.5 ng/mL upper limit of normal for PCT).

In conclusion, in the present study, serum levels of B₂–m and PCT in CHD patients were significantly, positively, and linearly correlated. This finding probably means that the presence of infection, as judged by PCT increases, enhances B₂–m production, aggravating the problem of DRA in these patients.